ポスター発表 (Poster Sessions)

情報伝達とその調節 Signal Transduction and Modulation
P1-1-28 光遺伝学的制御により明らかにされた、神経細胞におけるPIP₃シグナルの2つの緩衝機構 Optogenetic Control of PIP₃ Reveals Its Two Signal Buffering Mechanisms in Neurons ○角元利行^1,2, 中田隆夫^1,2 ○Toshiyuki Kakumoto^1,2, Takao Nakata^1,2 東京医科歯科大学医歯学総合研究科細胞生物学¹, 東京医科歯科大学脳統合機能研究センター² Dept. of Cell Biology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University¹, The Center for Brain Integration Research, Tokyo Medical and Dental University² Cells respond to changing external environments using intracellular signaling molecules. Accumulation of an active signaling molecule causes reduction in signal sensitivity. Thus, there should be signal buffering systems, which immediately decrease signal concentration within cells. Here we show two independent buffering mechanisms of phosphatidylinositol 3,4,5-trisphosphate (PIP₃) in growth cones. First, we developed a phosphatidylinositol 3-kinase (PI3K) photoswitch which can produce PIP₃ at specific locations upon blue light exposure. By using the PI3K photoswitch, we succeeded in locally producing PIP₃ in neurons for the first time. Local production of PIP₃ induced an expansion of the growth cone through the formation of dynamic actin-related structures such as lamellipodia and filopodia. However, the increase in PIP₃ concentration in the growth cone's plasma membrane was unexpectedly restricted, partially due to the increase in growth cone area. In addition, endocytosis of PIP₃-containing membrane selectively retrieved PIP₃ from the plasma membrane. Rac1 increased growth cone area, where the amount of PIP₃ was increased but PIP₃ concentration was unchanged. We also found that PIP₃ concentration did not much vary among neurites during neuronal polarization. The present study shows that two physical buffering mechanisms diminish PIP₃ signaling in a timescale of minutes.	P1-1-29 NAP-22はカルシニューリンの酵素活性を制御する Regulatory effect of NAP-22 on the activity of calcineurin ○小林優美¹, , 熊ノ郷晴子², 森田光洋¹, 中村俊³, 前川昌平¹ ○Yuumi Kobayashi¹, Ronan V da Silva¹, Haruko Kumanogoh², Mitsuhiro Morita¹, Shun Nakamura³, Shouhei Maekawa¹ 神戸大学大学院　理学研究科　生物学専攻¹, 国立精神・神経医療研究センター　神経研究所², 東京農工大学工学部³ Dept Biology, Graduate School of Kobe-Univ, Kobe¹, Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo², Faculty of Technology, Tokyo University of Agriculture and Technology, Tokyo³ NAP-22 (CAP-23, BASP1) is a neuron-enriched membrane protein localized in the presynaptic membrane regions such as the synaptic plasma membrane and the synaptic vesicles. This protein is also a major component of the neuronal lipid raft. Recent studies however showed that some amount of NAP-22 is present in the cytoplasm and in the nucleus. In order to know NAP-22 interacting proteins in the soluble fraction of rat brain, a pull-down assay using anti-NAP-22 antibody bound Sepharose-beads precharged with NAP-22 was performed and calcineurin(CaN) was identified in the precipitate. CaN is a Ser/Thr-phosphatase broadly distributed within neurons and its activity is enhanced with Ca²⁺-calmodulin. The participation of CaN in the processes of LTP and LTD is well studied. Protective effect of CaN on neurons through the dephosphorylation of tau in Alzheiner's disease is also considered. Identification of the CaN binding proteins is hence of primary importance. The binding of these proteins was confirmed using bacterially expressed CaN and brain-derived NAP-22. NAP-22 decreased the phosphatase activity of CaN on pNPP and this effect was prominent in the absence of Ca²⁺-calmodulin. Co-localization of these proteins at the presynaptic region was shown using immunostaining of cultured neurons. These results suggest that NAP-22 participates in the cell-signaling pathways through the regulation of the activity and localization of CaN.
P1-1-30 Dock6は脊髄後根神経節ニューロンの軸索伸展を制御する Effects of the in vivo knockdown of Dock6 on dorsal root ganglion neurons ○宮本幸¹, 鳥居知宏¹, 田上昭人¹, 山内淳司^1,2 ○Yuki Miyamoto¹, Tomohiro Torii¹, Akito Tanoue¹, Junji Yamauchi^1,2 国立成育医療センター研究所　薬剤治療研究部¹, ヒューマンサイエンス振興財団² Dept. Pharmacol., NICHD (JAPAN), Tokyo¹, The Japan Human Health Sciences Foundation, Tokyo, Japan² Axon morphogenesis, which includes neurite outgrowth, axon guidance, branching, and synapse formation, is an orchestrated developmental process eventually leading to the establishment of the neuronal circuits in mammals. Peripheral nervous system (PNS) development in mammals is unique in two ways. First, an interaction between two different cell types, namely neurons and Schwann cells, is responsible for part of PNS development. Second, sensory neurons such as dorsal root ganglion (DRG) neurons exhibit comparatively long axons. The mechanisms underlying sensory axon navigation are mediated by signaling molecules controlling continuous and complex cytoskeletal changes. Rho GTPases are one class of signaling molecules that control such changes. Yet there remain missing links in the pathway coupling extracellular signals to Rho GTPases. We previously characterized Dock6 as an atypical Dock180-related guanine-nucleotide exchange factor for Rac and Cdc42. To investigate the role of Dock6 in DRG neurons, we generated genetically-modified mice for Dock6. We injected the DNA construct encoding Dock6 shRNA into mouse fertilized eggs and generated Dock6 shRNA transgenic mice. They displayed an effective knockdown of Dock6 protein in DRG neurons. Dock6 shRNA transgenic mice exhibited a phenotype characterized by shortened peripheral neuronal fibers, which extend from the ganglia to the ventral roots, compared with those seen in control nontransgenic littermates, suggesting the involvement of Dock6 in the proper formation of peripheral neuronal fibers. Also, in DRG neurons isolated from Dock6 shRNA transgenic mice resulted in a decrease in axon length, axon number, and each axon's branch points. Together, these results suggest that Dock6 plays a key role in axon extension in PNS. Further studies on the activation mechanisms of Dock6 would promote our understanding of its role not only in axon morphological changes but also in interactions with Schwann cells.	P1-1-31 NAP-22 と脳のグルタミン酸デカルボキシラーゼ(GAD)の相互作用 Interaction of NAP-22 with brain glutamic acid decarboxylase (GAD) ○前川昌平¹, 小林優美¹, 小田垣真一¹, 牧野碧¹, 熊ノ郷晴子², 中村俊³, 森田光洋¹, 林文夫¹ ○Shohei Maekawa¹, Yuumi Kobayashi¹, Shin-Ichi Odagaki¹, Midori Makino¹, Haruko Kumanogoh², Shun Nakamura³, Mitsuhiro Morita¹, Fumio Hayashi¹ 神戸大学大学院理学研究科生物学専攻¹, 国立精神・神経研究センター神経研究所², 東京農工大学³ Dept. Biol. Graduate School of Science, Kobe-University¹, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan², Tokyo University of Agriculture and Technology, Koganei, Japan.³ NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched protein localized mainly in the synaptic vesicles and the synaptic plasma membrane and one of the main proteins recovered in the detergent-resistant low-density membrane microdomain fraction prepared from brain. In order to understand the physiological function of NAP-22, its binding proteins in the soluble fraction of rat brain were screened with a pull-down assay. Glutamic acid decarboxylase(GAD) was detected through LC-MS/MS, and western blotting using a specific antibody confirmed the result. Two isoforms of GAD, GAD65 and GAD67, were expressed in bacteria as GST-fusion forms and the interaction with NAP-22 was confirmed in vitro. Partial co-localization of NAP-22 with GAD65 and GAD67 was also observed in cultured neurons. The binding showed no effect on the enzymatic activity of GAD65 and GAD67. These results hence suggest that NAP-22 could participate in the transport of GAD65 and GAD67 to the presynaptic termini and their retention on the synaptic vesicles as an anchoring protein.
P1-1-32 アメフラシ口球神経節におけるD-アミノ酸の摂食運動パターン形成への影響 Effects of D-amino acids on central pattern generator of Aplysia buccal ganglion ○木村亮介¹, 吉見靖男¹, 長濱辰文² ○Ryosuke Kimura¹, Yasuo Yoshimi¹, Tatsumi Nagahama² 芝浦工大院・理工・応用化学¹, 東邦大・薬² Dept. Appl. Chem., Shibaura Inst. Technol., Tokyo, Japan¹, Dept. Biophys., Fac. Pha. Sci., Toho Univ., Funabashi, Japan² Most of amino acids are optically active, and then have enantiomers. It has been believed for long time that D- amino acids are useless in biological activity. However, recent researches using enantioselective analytical technologies demonstrate that D-amino acids play an important role in mammalian central nervous systems (CNS). Then an analysis of physiological functions of D- amino acids in CNS is thought to contribute to the development of therapy for brain disorder such as schizophrenia.In this work, the central pattern generator (CPG) for feeding in Aplysia buccal ganglion was driven by repetitive electrical stimulation of the esophageal nerve and the effects of D-Serine (D-Ser) on the CPG output were explored. The output was detected from intracellular recording of the rhythmic bursts of spikes in B3 neuron. After administration of 200 μM D-Ser, the frequency of rhythmic bursts increased to 1.5 times, and the duration of each burst was reduced by half compared with the control values. These values recovered by washing with artificial seawater. At the last annual meeting, we reported that the burst frequency of B3 neuron during the rhythmic responses of the same CPG outputs decreased by administration of D-amino acid oxidase which denatures D-Ser. These results strongly indicate that D-Ser plays an important role in decision of the CPG output in Aplysia buccal ganglion.	P1-1-33 素子数が規定された培養神経細胞ネットワークにおける自発的神経活動の解析 Emergence of spontaneous network activity from micropatterned neuronal networks in culture ○山本英明^1,2, 森田麻裕³, 出村崇徳³, 河野翔³, 谷井孝至^2,3, 中村俊¹ ○Hideaki Yamamoto^1,2, Mayu Morita³, Takanori Demura³, Sho Kono³, Takashi Tanii^2,3, Shun Nakamura¹ 東京農工大・工・生命工¹, 早大・ナノテク², 早大・基幹理工・電子光システム³ Dept of Biotechnol & Life Sci, Tokyo Univ of Agricul & Technol, Tokyo¹, Nanotechnol Res Ctr, Waseda Univ, Tokyo², Dept of Elec & Photonic Sys, Waseda Univ, Tokyo³ Rat cortical neurons develop to form functional networks in culture. After several days of culture, the neurons begin to produce network-driven spontaneous activity, characterized by highly synchronized bursts of action potentials. Spontaneous neural activity in vivo has been shown to be important in both neural development and cortical information processing, but the precise mechanism as to how the rhythmic activity spontaneously emerges without any external stimuli remains unclear. Here we leveraged surface micropatterning techniques to control the number of neurons and extent of their axon/dendrite elaboration in a given network of cultured neurons. Using this micropatterned neuronal network, we studied how spatiotemporal pattern of spontaneous network bursts (sNBs) depend on the number of cells and the excitatory-to-inhibitory (E-I) balance in a given network to investigate the generating mechanisms sNBs. Micropatterned surface was prepared using electron-beam lithography by patterning cell-permissive poly-lysine islands in a non-permissive background of polyethylene glycol silanes. Dimensions of the permissive islands used in this experiment were: 200×200 μm², 500×500 μm², and 1000×1000 μm². Embryonic rat cortical neurons were cultured on the micropatterned surface for 10-11 days, and activity of the network was analyzed by multicellular calcium imaging. Analysis of sNB in networks of various pattern sizes showed that frequency of sNB decreases with decreasing number of cells in a network. Yet surprisingly, we found that a network with only 12 cells on a 200×200-μm² island exhibited sNB. The number of cells in the network is four times smaller than that previously proposed to be required for generation of sNB in cultured neurons. Role of E-I balance in the cultured network is also being studied by post-hoc immunostaining of the cells, and these results will also be presented.
P1-1-34 免疫電子線トモグラフィーによるNGF刺激した分化PC12細胞の神経突起バリコシティーにおける神経栄養因子受容体TrkAの三次元分布 Three-dimensional distribution of TrkA neurotrophin receptors in neurite varicosities of differentiated PC12 cells treated with NGF determined by immunoelectron tomography ○西田倫希¹, 吉村亮一², 遠藤泰久² ○Tomoki Nishida¹, Ryoichi Yoshimura², Yasuhisa Endo² 阪大・超高圧電顕センター¹, 京都工繊大・院・応用生物² Research Center For Ultra-High Voltage Electron Microscopy Osaka University, Osaka, Japan¹, Division of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan² Nerve growth factor (NGF) initiates the activation of TrkA tyrosine kinase receptors and numerous subsequent signaling cascades. However, the dynamics of the process including the translocation of TrkA is still unclear. In this study, the effect of NGF on the endocytic process and TrkA localization in the neuronal cell line PC12 was analyzed by live-cell imaging and immunoelectron tomography using an ultra-high voltage electron microscope (UHVEM). NGF re-stimulation enhanced the endocytic uptake of the fluorescent indicator into acidic organelles within varicosities as well as cell bodies. NGF also significantly increased the number of TrkA-containing varicosities. Immunoelectron tomography in whole-mounted cells showed that NGF induced the recruitment of TrkA to the surface membrane of neurite varicosities as well as the multivesicular bodies (MVBs) and lysosomal complexes inside the varicosities. Three-dimensional analysis revealed that invagination pits and intralumenal vesicles of MVBs contained TrkA immunoreactivity. In addition, TrkA immunoreactivity was scattered in the lysosomal matrices after NGF treatment. These results suggest that the neurite varicosities are intensely active in intracellular membrane trafficking and play an important role in the degradation and accumulation of the NGF receptor, TrkA, after ligand stimulation.	P1-1-35 IP₃/Ca²⁺シグナルによるシナプス内GABA-A受容体の側方拡散およびシナプスでの集散の制御 Regulation of synaptic GABA-A receptor lateral mobility and clustering by IP₃/Ca²⁺ signaling ○丹羽史尋¹, 坂内博子¹有薗美沙¹, 宮本章歳¹, 杉浦琴美¹御子柴克彦¹ ○Fumihiro Niwa¹, Hiroko Bannai¹, Sherwood Mark¹, Misa Arizono¹, Akitoshi Miyamoto¹, Kotomi Sugiura¹, Sabine Levi^2,3, Antoine Triller³, Katsuhiko Mikoshiba¹ 理研・BSI・発生神経生物¹, Institut du Fer a Moulin・Inserm², IBENS・Inserm³ Laboratory for Developmental Neurobiology, Brain Science Institute (BSI), RIKEN, Wako¹, Institut du Fer à Moulin, Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche-S 839, Université Pierre et Marie Curie, Paris², Institut de Biologie de l'Ecole Normale Supérieure, Institut National de la Santé et de la Recherche Médicale U1024, Centre National de la Recherche Scientifique UMR8197, Paris³ The number of GABA-A receptors (GABA_AR) clustering at the inhibitory synapse is a crucial determinant of GABAergic synaptic transmission efficacy. Ca²⁺ influx in response to excitatory neuronal activity results in the dispersal of synaptic GABA_AR clusters. In addition to Ca²⁺ influx, Ca²⁺ release from the intracellular Ca²⁺ store also plays various roles in neurons. However, the impact of Ca²⁺ release on the inhibitory synapse remains to be elucidated. Here, we examined whether the inositol 1, 4, 5-trisphosphate (IP₃)-induced Ca²⁺ release (IICR) is involved in the regulation of synaptic clustering and membrane dynamics of GABA_ARs. In hippocampal neurons from mice lacking intracellular Ca²⁺ releasing channel IP₃ receptor type 1 (IP₃R1), clusters of GABA_AR and those of its scaffold protein gephyrin were significantly smaller than in wild type neurons. This effect was not accompanied with reduced amount of GABA_ARs on the cell surface. Single particle tracking with quantum dot revealed that the loss of IP₃R1 and inhibition of IICR enhanced the lateral diffusion of GABA_ARs. The increase in GABA_AR lateral mobility resulting from the loss of IICR was completely prevented by cyclosporine A, an inhibitor for calcineurin. Our results suggest that IP₃/Ca²⁺ signaling contribute to the stabilization of synaptic GABA_AR clusters through the regulation of GABA_AR lateral diffusion, possibly through phosphorylation.
P1-1-36 Gタンパク質共役型受容体GPCR活性化によるCa²⁺/カルシニューリン経路を介した活動依存的な遺伝子発現制御系に関する解析 G-protein-coupled receptor-induced activity-dependent gene expression through a Ca²⁺/calcineurin pathway in neurons ○津田正明¹, 福地守¹, 高崎一朗², 竹森洋³, 田渕明子¹ ○Masaaki Tsuda¹, Mamoru Fukuchi¹, Ichiro Takasaki², Hiroshi Takemori³, Akiko Tabuchi¹ 富山大院医薬分子神経生物¹, 富山大・生命セ・遺伝子実験施設², 医薬基盤研究所・代謝疾患関連タンパク探索プロジェクト³ Dept Biol Chem, Grad Sch of Med & Pharm Sci, Univ of Toyama, Toyama¹, Div Mol Gen Res, Life Sci Res Ctr, Univ of Toyama, Toyama², Laboratory of Cell Signaling and Metabolism, National Institute of Biomedical Innovation³ Although coordinated intracellular signals evoked via excitatory and modulatory synaptic transmissions with an induction of immediate-early genes (IEGs) are needed for motivation and memory, the intracellular mechanisms underlying this coordination are still unclear. Because activation of G-protein-coupled receptors (GPCRs) potentiates the activity of the NMDA receptor (NMDA-R), we investigated how IEGs are induced by stimulating GPCRs. Signal transduction via the NMDA-R's potentiation induced by stimulating PAC1, a Gs- and Gq-coupled GPCR which is a receptor for pituitary adenylate cyclase-activating polepeptide (PACAP), differed from that triggered by direct activation of the NMDA-R with NMDA, due to the selective usage of a Ca²⁺/calcineurin (CN) pathway, resulting in the efficient mRNA expression of CN-regulated IEGs, including Bdnf, through CRTC1/CREB-mediated transcription. This Ca²⁺/CN pathway was activated not only by selective stimulation of Gs- or Gq-coupled GPCRs but also by direct activation of the PKA or PKC pathway. This GPCR-induced gene expression may operate under conditions of coordinated synaptic transmission in neurons, possibly irrespective of the kind of GPCR.	P1-1-37 cAMPによるオートファジーの制御 Regulation of autophagy by cAMP ○井上宏子¹, 樋口貴大¹ ○Hiroko Inoue¹, Takahiro Higuchi¹ 早大・先進理工・電気・情報生命¹ Waseda Univ. Tokyo¹ Autophagy is a degradation pathway for cytoplasmic proteins and organelles in eukaryotes. Autophagy leads to cell death (type II programmed cell death) under certain circumstances, and plays a cytoprotective role in maintaining cell viability to avoid cell death. Although the mechanisms of autophagy regulation are not completely understood, it has been shown that the target of rapamycin (TOR) signaling pathway plays a major role in controlling autophagy induction. TOR is a Ser/Thr protein kinase that regulates cell growth and proliferation, and its activity is inhibited by nutrient starvation, which has been shown to be a crucial step for autophagy induction. On the other hand, TOR-independent pathways are also suggested to regulate autophagy. The cAMP signaling pathway has been shown to control autophagy in yeast and mammalian cells. To evaluate the involvement of the cAMP in autophagy induction, we examined effect of forskolin on autophagy in mammalian cells. Forskolin elevated LC3-II level in rodent neuronal NG108-15 cells, but not in mouse embryonic fibroblasts. LC3-II expression was augmented by addition of lysosomal inhibitor chloroquine in NG108-15 cell. Therefore, it is probable that cAMP induces autophagy in some cells, but the effect is different in different cell types. We also investigated whether PKA was involved in cAMP-induced autophagy. Upregulation of LC3-II expression by forskolin was not affected by membrane-permeable PKA inhibitor peptide PKI (14-22). Furthermore, phosphorylation level of S6K1, a substrate of mTOR was decreased in response to forskolin treatment. Taken together, cAMP induced autophagy dependently of mTOR in NG108-15 cells, but PKA activation was not involved in the autophagy.
P1-1-38 ミクログリア細胞のP2X7受容体活性化を介するRunx2発現上昇 Upregulation of Runx2 expression through activation of P2X7 receptors in microglial cells ○中里亮太¹, 宝田剛志¹, 米田幸雄¹ ○Ryota Nakazato¹, Takeshi Takarada¹, Yukio Yoneda¹ 金沢大院・薬・薬物学¹ Laboratory of Molecular Pharmacology, Kanazawa University Graduate School, Kanazawa, Japan¹ We have previously shown the functional expression of the master regulator of osteogenesis and chondrogenesis, runt related transcription factor-2 (Runx2), by microglial cells, along with a transient increase in Runx2 mRNA expression during sustained exposure to 1 mM ATP in a manner mediated by the P2X7 receptor subtype. In this study, we further investigated the mechanisms underlying upregulation of Runx2 expression in microglia cells exposed to ATP in association with evaluating the significance in microglial functions. A similarly significant increase was seen in Runx2 mRNA and corresponding protein in mouse primary cultured microglia exposed to 1 mM ATP for 6 to 12 h. The increase by ATP was significantly prevented for Runx2 mRNA expression after prior incubation with 10 μg/ml cyclosporine A and 10 μg/ml FK-506 in murine microglial cell line BV-2 cells, but not by that with 10 μM U0126, 10 μM SB203580, 5 μM MG132 or 10 μM rapamycin. BV-2 cells were transiently transfected with siRNA for Runx2, followed by further culture for an additional 24 h and subsequent exposure to 1 mM ATP for different periods. Although Runx2 siRNA significantly decreased Runx2 protein expression, Runx2 siRNA alone failed to markedly affect the number of microglial cells with processes. However, exposure to ATP markedly reduced the number of cells with at least one process in BV-2 cells with Runx2 siRNA 6 to 24 h after exposure. These results suggest that extracellular ATP would selectively up-regulate the expression of Runx2 endowed to promote microglial processes through a mechanism relevant to the activation of calcineurin in microglial cells.	P1-1-39 成熟脳における細胞質型チロシンホスファターゼShp2の機能解析 Functional analysis of a non-receptor type protein tyrosine phosphatase Shp2 in the adult brain ○草苅伸也¹, 齋藤文仁², 橋本美穂¹, 柴崎貢志³, 吾郷由希夫⁴, 松田敏夫⁴小谷武徳⁶, 村田陽二⁶, 的崎尚⁶, 大西浩史¹ ○Shinya Kusakari¹, Fumihito Saitow², Miho Sato-Hashimoto¹, Koji Shibasaki³, Yukio Ago⁴, Toshio Matsuda⁴, Benjamin G. Neel⁵, Takenori Kotani⁶, Yoji Murata⁶, Takashi Matozaki⁶, Hiroshi Ohnishi¹ 群馬大学・生調研・バイオシグナル¹, 日医大・薬理学², 群馬大・院・医・分子細胞生物³, 大阪大・院・薬・薬物治療⁴, オンタリオがん研究所⁵, 神戸大・院・医・シグナル統合⁶ Lab Biosig Sci, IMCR, Gunma Univ, Gunma¹, Dept Pharmacol, Nippon Med Schl, Tokyo², Dept Mol Cell Neurobiol, Gunma Univ Grad Schl Med, Gunma³, Lab Med Pharmacol, Grad Schl Pharm Sci, Osaka Univ, Osaka⁴, Ontario Cancer Inst, Canada⁵, Div Mol Cell Signal, Dept Biochem Mol Biol, Kobe Univ Grad Schl Med, Kobe⁶ A non-receptor type protein tyrosine phosphatase, Shp2 (Src homology 2-containg protein tyrosine phosphatase 2), acts as a positive regulator of Ras-MAPK cascade downstream of growth factor receptors, and thereby participates in cell proliferation and differentiation. However, Shp2 is also expressed in post-mitotic neurons in the adult brain. To investigate the physiological roles of Shp2 in matured neurons, we selectively deleted Shp2 in post-mitotic forebrain neurons by crossing CaMKII-Cre transgenic mice with Shp2-floxed mice. Resulting Shp2 mutant mice (CaMKII-Cre: Shp2^flox/flox) exhibited increased locomotor activity and reduced anxiety-like behavior. Behavioral sensitivity of these mice to methylphenidate (MPH), a drug that increases extracellular monoamines in the brain, was normal when compared with that of wild-type control mice. Consistently, MPH induced an increase in the concentration of extracellular monoamines in the striatum of the mutant mice to the same extent as in wild-type control. In contrast, novelty-induced expression of immediate early genes in the brain was significantly reduced in the mutant mice, suggesting a possible reduction of neuronal activity. Furthermore, abnormal slow waves in electroencephalograms, which are often associated with impaired brain functions, were observed in the mutant mice. Electrophysiological analyses showed that post-tetanic potentiation, a form of short-term synaptic plasticity, was markedly attenuated in the hippocampal slice prepared from the mutant mice. We also found that knockdown of Shp2 in cultured neurons significantly reduced BDNF-induced activation of MAPK. Our data suggest a possible involvement of Shp2 in regulation of neural excitability and/or synaptic plasticity that influence animal behaviors. BDNF signaling may be an important mechanism for such functions of Shp2.
P1-1-40 強制水泳ストレスを受けたマウスの海馬におけるシグナル分子のリン酸化状態変化に対する低体温の影響 Hypothermia-dependent and -independent effects of forced swim on the phosphorylation states of signaling molecules in mouse hippocampus ○橋本美穂⁵, 林由里子^1,2, 草苅伸也¹, 浦野江里子¹, 滋野雅大³, 関島恒夫³, 小谷武徳⁴, 村田陽二⁴, 村上博和², 的崎尚⁴, 大西浩史⁵ ○Miho Hashimoto⁵, Yuriko Hayashi^1,2, Shinya Kusakari¹, Eriko Urano¹, Masahiro Shigeno³, Tsuneo Sekijima³, Takenori Kotani⁴, Yoji Murata⁴, Hirokazu Murakami², Takashi Matozaki⁴, Hiroshi Ohnishi⁵ 群馬大・生調研・バイオシグナル¹, 群馬大・院・保・生体情報検査², 新潟大・院・自然科学・環境共生³, 神戸大・院・医・シグナル統合⁴, 群馬大・院・保・生体情報検査⁵ Lab Biosig Sci, IMCR, Gunma Univ, Gunma¹, Dept Lab Sci, Gunma Univ Grad Schl Health Sci, Gunma², Dept Environ Sci Tech, Grad Schl Sci and Tech, Niigata Univ, Niigata³, Div Mol Cell Signal, Dept Biochem Mol Biol, Kobe Univ Grad Schl Med, Kobe⁴, Dept Lab Sci, Gunma Univ Grad Schl Health Sci, Gunma⁵ Forced swim stress induces diverse biochemical responses in the brain of rodents. Here, we examined the effects of hypothermia induced by forced swim in cold water on the phosphorylation of forced swim-sensitive signaling molecules in the mouse brain. Forced swim in cold water (23°C) induced a significant increase in the level of tyrosine phosphorylation of SIRPα, a neuronal membrane protein, in mouse hippocampus, while such effect of forced swim was markedly reduced in mice subjected to forced swim in warm water (37°C). Forced swim in cold water also induced phosphorylation of mitogen-activated protein kinase kinase (MEK) as well as of cAMP response element-binding protein (CREB), or dephosphorylation of α isoform of Ca²⁺/calmodulin-dependent protein kinase II (αCaMKII) in the hippocampus. These effects of forced swim on the phosphorylation of MEK, CREB, and αCaMKII were also lost in mice subjected to forced swim in warm water. Genetic ablation of SIRPα did not change the phosphorylation states of these molecules in the brain. Forced cooling of anesthetized mice, which induced a marked increase in the phosphorylation of SIRPα, induced dephosphorylation of αCaMKII in the brain, while the same treatment did not affect the phosphorylation level of MEK and CREB. Hibernation also induced an increase and a decrease of the phosphorylation of SIRPα and αCaMKII, respectively, in the brain of chipmunk. These results suggest that hypothermia is a major element that determines the levels of phosphorylation of αCaMKII and SIRPα during the forced swim in cold water, while it is not for the phosphorylation levels of MEK and CREB.

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